No abstract

Human IgA is both a major serum immunoglobulin and the most abundant antibody class in seromucous secretions (1). The mucosal surfaces bathed by these secretions, such as those of the respiratory, gastrointestinal, and genitourinary tracts, are major potential sites of invasion due to their vast surface area. IgA therefore serves as a key first line of defense against many invading pathogens. Like all antibodies, IgA is capable of both recognizing the foreign invader and triggering its elimination. The latter process is frequently mediated by the interaction of the Fc region of IgA with Fc␣ receptors (Fc␣R) 1 present on the surface of neutrophils, macrophages, monocytes, and eosinophils (2, 3). The human myeloid Fc␣R, CD89, possesses two extracellular Ig-like domains and displays homology to the three classes of human IgG Fc receptors (Fc␥RI, Fc␥RII and Fc␥RIII) and the high affinity IgE receptor Fc⑀RI, albeit at a lower level than these receptors do to each other (4). Interaction of CD89 with IgA, aggregated either by binding to antigen or artificially, acts as a potent trigger for an array of myeloid cell functions including phagocytosis, antibody-dependent cellmediated cytotoxicity, superoxide generation, enzyme and inflammatory mediator release, and clearance of immune complexes (3). A detailed understanding of the molecular basis of the IgA-Fc␣R interaction is clearly important if the increasingly appreciated potential of recombinant IgA in numerous therapeutic applications (5-7) is to be fully realized.Although others have described expression of human IgA in insect (8) and plant cells (5), we have expressed hapten-specific recombinant human IgA of both subclasses, IgA1 and IgA2, in mammalian cell hosts (9 -11). Here, we have used an extensive panel of chimeric and site-directed mutant IgAs expressed in CHO K1 cells to identify residues critical for Fc␣R binding. We have constructed domain swap antibodies through exchange of the homologous C-terminal CH3 domains between human IgA1 and IgG1 in order to ascertain the contribution of each Fc domain to the interaction with Fc␣R. An IgA1 lacking the C-terminal 18 amino acid tailpiece has also been assayed for ability to bind the receptor. To allow more precise localization of the interaction site, a number of IgA1 mutants with single substitutions in loop regions lying at the interface of the CH2 and CH3 domains have been generated. The effects of such mutations on the ability to bind Fc␣R are consistent with the interdomain region of the Fc playing a critical role in binding to the receptor. Further support for this proposal is lent through correlation of the binding ability of IgAs derived from other species with sequence differences in these loops.To more readily assess receptor interaction, we have developed stable CHO cell transfectants expressing high levels of CD89, which have allowed, for the first time, comparison of the relative binding affinities of the different IgA molecules. As an additional, more physiologically relevant test for function, we hav...

No abstract

There are only a few proteins identified at the cell surface that could directly regulate plant cell wall functions. The cell wall-associated kinases (WAKs) of angiosperms physically link the plasma membrane to the carbohydrate matrix and are unique in that they have the potential to directly signal cellular events through their cytoplasmic kinase domain. In Arabidopsis there are five WAKs and each has a cytoplasmic serine/threonine protein kinase domain, spans the plasma membrane, and extends a domain into the cell wall. The WAK extracellular domain is variable among the five isoforms, and collectively the family is expressed in most vegetative tissues. WAK1 and WAK2 are the most ubiquitously and abundantly expressed of the five tandemly arrayed genes, and their messages are present in vegetative meristems, junctions of organ types, and areas of cell expansion. They are also induced by pathogen infection and wounding. Recent experiments demonstrate that antisense WAK expression leads to a reduction in WAK protein levels and the loss of cell expansion. A large amount of WAK is covalently linked to pectin, and most WAK that is bound to pectin is also phosphorylated. In addition, one WAK isoform binds to a secreted glycine-rich protein (GRP). The data support a model where WAK is bound to GRP as a phosphorylated kinase, and also binds to pectin. How WAKs are involved in signaling from the pectin extracellular matrix in coordination with GRPs will be key to our understanding of the cell wall's role in cell growth.